A thorough understanding of spermatogenesis is important for advances in male infertility research and male contraceptive development. The long-term goal of this research is to understand how posttranscriptional regulatory mechanisms control the expression of sperm proteins. The translational repression of some mRNAs at specific periods during spermatogenesis is a key strategy that is used in plants, insects and mammals. Recent genetic and molecular studies of spermatogenesis in the fruit fly Drosophila have identified three components of a translational repression pathway. These are the RNA binding protein TSR, the Scpr mRNA that it regulates, and the GTP-binding protein ARF, which binds TSR and may modulate its function. With this information, it is now possible to address fundamental questions about the mechanism of translational regulation, using a combination of genetic, molecular and biochemical approaches. The first specific aim of this proposal focuses on a detailed characterization of the TSR-Scpr mRNA interaction, and how Scpr mRNA becomes activated for translation at the appropriate time. In vitro binding studies will be used to identify the sequences required for the interaction, and their biological relevance will be tested in transgenic flies. The second aim is to generate mutations in ARF and study its function in vivo during spermatogenesis. The effects of these mutations on Scpr and TSR will also be assayed. The final specific aim will address the question of how the TSR-ARF protein-protein interaction is regulated, and the effect of that interaction on the subcellular localization of TSR. The yeast two-hybrid system will be used with wild type and mutant proteins to define the role of guanine nucleotides in the interaction and the regions of TSR that are important for protein binding. Additional experiments will use immunoelectron microscopy to examine the localization of TSR and ARF in developing spermatocytes and spermatids. Given the fact that human testis proteins related to Scpr have important roles in spermatogenesis, the results of the proposed research should not only provide fundamental knowledge regarding translational regulation, but also provide novel molecular targets for modulating sperm production in humans.